Microbial Growth Kinetics Lecture 2

Fermentation Process

Types of Culture Fermentation - carried out as batch, continuous and fed-batch Dictated largely by type of product being produced Mode of operation of fermentation is largely governed by the type of product being produced.

Batch Culture Closed culture system – initially contains limited amount of nutrient No growth – lag phase – time of adaptation Growth rate increases – grow constantly at maximum rate – Log or exponential phase The inoculated culture will pass through a number of phases. In commercial processes lag phase must be reduced as much as possible and can be done by using a suitable inoculum

Batch Culture- Exponential phase/Trophophase Exponential phase equation: dx/dt = µx where x is the concentration of microbial biomass, t is time in hours and µ is the specific growth rate in hours Nutrients are in excess and organism is growing at its maximum specific growth rate = µmax

Batch Culture – Deceleration phase Cessation of growth – due to depletion of some essential nutrient in medium (substrate limitation) Accumulation of some autotoxic product of organism in medium (toxin limitation) Or combination of both Growth results in consumption of nutrients and excretion of microbial products – events which influence growth of organism. Thus after certain time the growth rate ceases.

Batch Culture – Stationary phase/Idiophase Point where growth rate has declined to zero. Population is metabolically active – producing secondary metabolites Maximum population phase Secondary metabolites are not produced during exponential phase. But in late exponential phase there is accumulation of lipid and carbohydrate

What is Yield Factor (Y)? It is a measure of efficiency of conversion of any one substrate into biomass. It is not constant – varies according to growth rate, pH, temperature, the limiting substrate and concentration of substrates in excess.

How can deceleration phase be tested? Zone A to B – increase in initial substrate concentration – proportionally increases biomass produced at stationary phase x=Y(Sr-s) where x is concentration of biomass produced Y is yield factor, Sr is initial substrate con. and s is residual conc. The nature of limitation of growth may be explored by growing the organism in presence of a range of substrate concentrations and plotting biomass concentration at stationary phase against initial substrate concentration. Over zone A to B, s equals zero at the point of cessation of growth. Over zone C to D an increase in initial substrate concentration does not give proportional increase in biomass. This may be due to either the exhaustion of another substrate or accumulation of toxic products. Over the zone B to C the utilization of substrate is deleteriously affected by either accumulating toxins or availability of another substrate

Growth kinetics of Metabolites Growth-linked products/Primary metabolites Formation of growth linked product is described by equation dp/dt = qpx where p is the concentration of the product qp is specific rate of product formation (mg product/g biomass/h) Product formation is related to biomass production by equation dp/dx = Yp/x Where Yp/x is the yield of product in terms of biomass (g product/g biomass) Formation of growth linked product is described by equation. Non-growth linked products/secondary metabolite

Batch fermentation – used for? Producing biomass – fastest growth rate and maximum cell population Primary metabolites – extend exponential phase Secondary metabolites – decreased growth in log phase Decreased growth rate in log phase allows for earlier secondary metabolite production

Continuous Culture (Chemostat) Addition of fresh medium to the vessel – exponential growth Medium is substrate limited Overflow device – added medium displaces equal volume of culture – continuous production of cells - Formation of new biomass balanced by loss of cells from vessel Exponential growth in batch culture is prolonged by addition of fresh medium to the vessel. The medium is substrate limited (by some component in the medium) and not toxin limited. Exponential growth will continue till the additional substrate is exhausted. If medium is continuously added to such a culture at a suitable rate then steady state is achieved by formation of new biomass by the culture that is balanced by loss of cells from the vessel.

Chemostat culture Chemostat culture – Cells and spent medium are continuously removed - State of culture is dependent upon flow rate of fresh medium

Continuous Culture (Chemostat) Dilution rate - The flow of medium into the vessel is related to volume of the vessel D = F/V - F is flow rate (dm3/h) - V is volume (dm3) D is dilution rate in per hour Net change in cell concentration over a time period = dx/dt = growth (µx) – output (Dx) In Continuous culture, specific growth rate is controlled by dilution rate which is an experimental variable. In batch cultures, an organism will grow at its maximum specific growth rate. In continuous cultures the culture may be operated only at dilution rates below the maximum specific growth rate. The growth of the cells in a continuous culture of this type is controlled by availability of growth limiting chemical component of the medium and the system is described as a chemostat

Problems - Chemostat Problems – Imperfect mixing and wall growth Imperfect mixing – increase in degree of heterogeneity in fermenter Wall growth – Organism adheres to inner surfaces of reactor – increases heterogeneity Limited by coating inner surfaces of vessel with Teflon Increase in degree of heterogeneity because some will receive nutrients in excess and others will be under severe limitation. Problem can be resolved by feedback systems

Feedback systems - Chemostat Internal feedback – Limiting exit of biomass External feedback – Subjecting effluent stream to biomass separation Internal – limiting exit of biomass from the chemostat such that biomass in effluent stream is less concentrated than in vessel. External – Subjecting effluent stream to biomass separation process such as sedimentation or centrifugation and returning portion of concentrated biomass to growth vessel

Perfusion/Turbidostat Perfusion culture - Medium is pumped continuously Cells are retained Becoming popular for large-scale production Attains high cell density Cell separator Continuous supply of nutrients and removal of waste products promotes cell growth to a greater extent than in batch culture. Cell separator separates cells from medium that is pumped out from culture. Concentration of cells in culture is kept constant by controlling the flow of medium such that the turbidity if culture is kept with certain narrow limits. This may be achieved by monitoring the biomass with a photoelectric cell and feeding the signal to a pump supplying medium to the culture such that the pump is switched on if the biomass exceeds the set point and is switched off if the biomass falls below the set point. The chemostat is more commonly used system because it has the advantage over turbidostat that former does not require complex control systems to maintain steady state. The latter is advantageous in continuous culture in avoiding washout of culture in its early stages.

Continuous culture Biomass production Microbial biomass produced for human or animal consumption – Single Cell Protein (SCP) Practice 1960s

What is Fed-batch culture? In open system/Fed-batch culture – involves controlled nutrient feeding Partial media changes at regular intervals (10 to the power of 6 cells/ml) Glucose can be added at daily intervals to increase final cell yield. Metabolites or metabolic products will accumulate to growth inhibitory concentrations

Fed-batch culture Initial batch cultures – fed continuously or sequentially with medium No removal of culture fluid Three types – Variable volume, Fixed volume and Cyclic fed-batch

Fed-batch culture Batch culture is fed in following ways Same medium + conc. used to establish batch culture is added –Variable volume Conc. solution of limiting substrate is added at a rate less than initial Very conc. Solution of limiting substrate at lesser rate than initial – Fixed volume Same medium used to establish batch culture is added resulting in an increase in volume

Cyclic fed-batch culture Life of variable fed-batch fermentation may be extended Withdrawing a portion of culture/residual culture Increase in dilution rate and specific growth rate Withdrawing a portion of culture/residual culture for further fed-batch rpocess. The decrease in volume results in significant increase in dilution rate (assuming that flow rate remains constant) and thus increase in specific growth rate.

Fed-batch culture Used for biomass Primary metabolite Secondary metabolite

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